Approach for sequencing glycosaminoglycan chains of proteoglycans.

摘要

The versatile biological activities of proteoglycans are mainly mediated by their glycosaminoglycan (GAG) components. Unlike proteins and nucleic acids, no satisfactory method for sequencing GAGs has been established. This thesis describes a fluorescent tag-based sequencing approach to analyze the GAG chains of two important proteoglycans, heparin and decorin.;The heparin proteoglycan has 15 GAG chains and is isolated only after proteolysis and partial degradation by endoglucuronidases. Approximately 10% of heparin still contains small remnants of core protein and thus is called peptidoglycan heparin. Heparin was labeled with a hydrophobic, fluorescent tag (N-(4-(6-dimethylamino-2-benzofuranyl) phenyl-isothiocyanate (NDBP)), to form NDBP-heparin. The NDBP-heparin was then enriched by chromatography on phenyl-Sepharose. A NDBP-linkage region tetrasaccharide was isolated by RP-HPLC from NDBP-heparin that had been treated with a mixture of heparin lyase I and heparin lyase III. The structure of NDBP-linkage region tetrasaccharide was determined to be ;The enriched NDBP-heparin was treated with lithium hydroxide to release heparin and tagged by reductive amination with 7-amino-1,3 naphthalene disulfonic acid (AGA), to form AGA-Xyl-heparin. AGA-Xyl-heparin was sequenced using heparin lyase I and heparin lyase III together with gradient PAGE. Thirteen saccharide residues from the core peptide have been characterized.;Decorin is unique because it contains a single GAG chain bound through a Xyl-O-Ser linkage. The GAG chain was released from the core protein by alkaline scission of the Xyl-O-Ser bond and subjected to reductive amination with AGA. The sequence of decorin was determined using similar approach for determining heparin sequence. Fifteen saccharide residues from the core peptide have been characterized.